Zernike 相衬 cryo-electron 显微镜揭示大鼠 TRPV4 阳离子通道的 3.5nm 结构。
A 3.5-nm structure of rat TRPV4 cation channel revealed by Zernike phase-contrast cryoelectron microscopy.
机构信息
Division of Nano-Structure Physiology, Okazaki Institute for Integrative Bioscience, Higashiyama, Myodaiji, Okazaki 444-8787 Japan.
出版信息
J Biol Chem. 2010 Apr 9;285(15):11210-8. doi: 10.1074/jbc.M109.090712. Epub 2009 Dec 31.
Abstract
The transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel responsive to various stimuli including cell swelling, warm temperatures (27-35 degrees C), and chemical compounds such as phorbol ester derivatives. Here we report the three-dimensional structure of full-length rat TRPV4 purified from baculovirus-infected Sf9 cells. Hexahistidine-tagged rat TRPV4 (His-rTRPV4) was solubilized with detergent and purified through affinity chromatography and size-exclusion chromatography. Chemical cross-linking analysis revealed that detergent-solubilized His-rTRPV4 was a tetramer. The 3.5-nm structure of rat TRPV4 was determined by cryoelectron microscopy using single-particle reconstruction from Zernike phase-contrast images. The overall structure comprises two distinct regions; a larger dense component, likely corresponding to the cytoplasmic N- and C-terminal regions, and a smaller component corresponding to the transmembrane region.
摘要
瞬时受体电位香草酸亚型 4（TRPV4）是一种非选择性阳离子通道，可响应多种刺激，包括细胞肿胀、温暖的温度（27-35°C）和化学化合物，如佛波酯衍生物。在这里，我们报告了全长大鼠 TRPV4 的三维结构，该结构从杆状病毒感染的 Sf9 细胞中纯化而来。六组氨酸标记的大鼠 TRPV4（His-rTRPV4）用去污剂溶解，并通过亲和层析和大小排阻层析进行纯化。化学交联分析表明，去污剂溶解的 His-rTRPV4 是四聚体。使用 Zernike 相差图像的单颗粒重建，通过冷冻电子显微镜确定了大鼠 TRPV4 的 3.5nm 结构。整体结构由两个不同的区域组成；较大的致密组件，可能对应于细胞质 N-和 C-末端区域，以及较小的组件对应于跨膜区域。
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